Bacteria and antimicrobial agents introduction 6.2:2:

introduction 1. 6.2:2:(1-X)

what are the main differences between Eukaryotes and Prokaryotes?

• Eukaryotes often bigger in size than Prokaryotes

• Prokaryotes don’t have a nuclear membrane or nuclei

• DNA in Prokaryotes is free-floating - in plasmids as well

• No membrane bound organelles in Prokaryotes e.g. mitochondria, EPR, golgi, chloroplast

• Flagella can be present in both

• Ribosome is smaller in Prokaryotes ~ 70s vs 80s

• prokaryotes often have single circular chromosome with no histones

• Prokaroytes divide by Binary fission

• Eukaroytes divide by mitosis

• Prokaryotes dont have sexual reproduction (they can transfer DNA) whereas Eukaryotes do by meiosis

• Prokaryotic cell wall contains peptidoglycan, fungi has chitin

• Prokaryotic plasma cell membrane lacks sterols unlike in eukaryotic 

what are common shapes of bactera?

• coccus - spherical

• coccobaciullus - cylinder type

• vibrio - thin and longand circular

• bacillus - longer than coccobacillus

• spirillium - spiral

• spirochete - higher frewuency of swirls

• e.g streptococci is 7 spheres connected as strept=7

What method do you use to distinguish between type of bacteria?

• Gram staining

What are the main types of bacteria?

• Gram positive

• Gram negative

• Acid-fast bacteria - not a type, basically a type of stain for specific bacteria

Explain the process of gram staining

• Heat fix specimen to slide

• flood slide with crystal violet solution and leave for 1 minute (purple)

• rinse the slide then flood with iodine solution for 1 minute

• Specimen will appear purple

• Rinse of excess iodine (orange)

• Declolourise with acetone for 5 seconds (depends on specimen) (clear)

• wash slide immediatley in water

• after washing, gram negative bacteria are not visible

• apply safranin counterstain for 30 seconds (red)

• wash in water, blot, and dry in air

• gram negative bacteria will be visible after adding this counterstain

Describe the result for Gram (+) during staining

• Staining depends on the ability of the bacteria to retain the dye

• Gram (+) have thick peptidoglycam cell wall

• Acetone dehydrates the cell wall and extracts the stain

• dehydration collapses the cell wall, closes the pores,  (causing it to intertwine) trapping the stain within the cells

• so dye remains in the cell = purple appearance

• so Gram (+) retains large crystal violet/iodine complex molecules upon acetone treatment

• in pic, cell wall is thicker than cell membrane

Why can antimicrobial agents enter gram (+) bacteria?

• Cell wall is porous, enzymes and antimicrobial agents enter the cell through here

• max size entry is 50,000 daltons

• most antimicrobial agents are <50,000 daltons

Give examples of Gram positive bacteria

• Staphylococcus - skin infections

• streptococcus - sore throat, dental caries

• streptomyces - source of antibodies

• Clostridium - wound infections

• bacillus

• lactobacillus

• listeria

• mycobacterium - leprosy, tuberculosis

• often cocci shape

Describe Gram (-) bacteria cell wall

• Thin peptidoglycan cell wall

• an additional outer membrane

• outer membrane contains porin proteins

• higher content of sterols/lipids in cell wall

Describe gram (-) staining result

• Acetone causes dehydration

• Acetone removes the outer membrane

• The thin cell wall peptidoglycan layer cannot prevent the removal of the crystal violet iodine complex

• As, in the thin cell wall, there is insufficient cell wall layer

• crystal violet/iodine complex washed away

• counterstain safranin stains it red as a lighter colour than purple

What do Porin channels do?

• allow diffusion of nutrients into the cell, into cytoplasm through membrane

• could be a drug target

Give examples of Gram (-) bacteria

• Neisseria Gonnorhoea & meningitis

• Brucella, Bordetella coughs and fevers

• Pseudomonas

• E.coli

• Salmonella

• shigella

• enterobacter

• V.cholera

• chlamidya

• campylobacter

• fusobacterium (anaerobic wound and dental infection)

• ureaplasma (urinary infections)

What do you use to stain acid-fast bacteria and why?

• Carbolfuschin (red)

• unlike gram (+) contains 60& lipid (mycolic acid) and less peptidoglycan - waxy prevents them from being stained

• so they are hard to stain, but don’t get discoulored by alcohol

Describe Mycobacteria (acid-fast) structure and growth

• thick cell wall

• additional capsule-like-material ontop

• unlike gram (+) contains 60& lipid (mycolic acid) and less peptidoglycan - waxy prevents them from being stained

• grow slowly as lipids restrict flow of nutrients and other agents into the cell

• lots of energy required to construct cell wall

• this is why Tuberculosis takes months to grow

give examples of acid fast bacteria

• mycobacterium tuberculosis (tuberculosis)

• mycobacterium leprae (leprosy)

what are the types of bacteria growth

• obligate aerobes

• facultative anaerobes

• obligate anaerobes e.g metronidazole in wounds- low oxygen

• aerotolerant anaerobes

• microaeophiles

what are sources of microorganisms?

• food

• water

• other humans

• environment

• animals, birds, insects, vectors

• human self microflora 

examples of zoonotic infections

give examples of common human microflora

• mostly in bowels as we know their used to break down bile acid, increase calcium absorption, makes vitamin B12, K, iron, calcium

what is a pathogen?

• an organism capable of causing disease

what is a true pathogen?

• causes disease in a healthy immunocompetent patient

what is pathogenicity

• ability to cause disease

• for example, neisseria gonorrhoeae requires pilli to attach to urethral epithelium

• strains with no pilli are non-pathogenic as they do not causes disease

• as washed away by urine

what is Virulences?

• an organisms relative power to causes disease

• illness can be asymptomatic, mild or very severe

• so virulence factors effect extent of disease

• a factor could be a human- how their immune system responds - human factors

give an example of pathogenicity vs virulence for understanding

• streptococcus pneumoniae does not causes disease if it lacks a capsule so isnon-pathogenic

• so the capsule determines the virulence of the organism 

• both pneumococci capsular type 3 and 30 produce capsules so are both pathogenic, however 3 causes severe disease whereas 30 rarely causes severe disease

• why? as capsule protects it against immune system

how do you identify that a bacterium is a pathogen?

• Koch’s Postulates

• the suspected pathogen must be present in all cases of the disease and absent from healthy animals - how? - by using microscope

• the suspected pathogen must be grown in pure culture - how? agar plate

• cells from a pure culture of the suspected pathogen must cause disease in a healthy animal - how? inoculate healthy animal

• the suspected pathogen must be re-isolated and shown to be the same as the original - how? - remove it from animal and observe under microscope

• antibodies to the organism appear during course of the disease

What are limitations to Koch’s Postulates

• it works well for many bacteria, but does not work for all infectious disease

• not all bacteria can be cultured in labs e.g. mycobacterium leprae

• ethics prohibit attempts to transfer from person to person so we use animals instead

• you need a pure sample, disease of possible polymicrobial origin wont be pure

• immunosupression may lessen the antibody response and render the host very susceptible to the disease - which may not be a good reflection

• genetic predisposition of individuals e.g if you have sickle cell, more likely to have certain infections

• hosts own flora - how do we know its not your own flora e.g. e.coli, candida spp.

what are the 5 characteristics of a successful pathogen?

• survival and transmission in the environment

• attachment to the surface of the host

• overcoming the body defences against infection

• ability to damage the host, directly or indirectly (disease)

• ability to replicate in the host, producing progency able to infect others (exit)

Give examples of strategies for controlling infectious disease

• water purification

• sanitation

• good levels of nutrition

• good living conditions (overcrowding)

• good personal hygiene 

• food- cold storage

• pasteurization

• food inspection and quality control

• adequate preparation and cooking e.g. meat

• control of vectors e.g. mosquitoes, rats, lices, ticks

• control of and contact with reservoir animals (rabies, bovine, TB, SARS)

• control measures e.g. lockdown

• vaccination

• antimicrobial chemotherapy

• education of risk

• screening programmes

• risk management strategies e.g. for covid or flu outbreak

what is antimicrobial chemotherapy?

• antimicrobial medicine e.g antibiotics

• depends on selectivity, so it must be selective

what must antimicrobials be, in order to be effective and what led to this discovery?

• must be toxic in vivo to the microbe and not the host

• use of dyes that differentially stained tryanosomes (protozoa) (stained the infective cells but not the actual human tissue) led to the concept of selective toxicity 

what is an antibiotic, and what is it made of?

• is a compound or derivative produced by a fungus or bacterium that kills or inhibits the growth of other microorganisms 

• can be synthetic

• can be natural e.g. by bacteria or fungi (penicllin)

how do we classify antibacterial agents?

• bacteriocidal or bacteriostatic - inhibits growth

• depends on the bacterial species e.g. chloramphenicol inhibits e.coli but kills haemophilus influenzae

• by target site

• by chemical structure 

why does bacteriostatic effect work even after medicine is complete?

• host immune system prevents the bacteriostatic bacteria from growing after

What are the antibacterial targets? EQ

• Cell wall

• cell membrane

• DNA/RNA

• folic acid synthesis

• protein synthesis on ribosomes

what do you need to know about antibacterial agents?

• what it is - chemical structure and synthesis

• what it does - mechanism, target

• where does it go - ADME

• when its used

• limitations - e.g. toxicit, efficacy, SE, CI

• cost - more expensive, less? - just an idea

examples of common antibacterial agents

what is spectrum of activity?

• which organisms are antimicrobials active against , so could be broad

How are antibiotics different from other drugs?

1. activity is not directed at human metabolic process - target is the microorganism

2. activity is needed against several body sites

3. used in large numbers of patients for a short period

4. used prophylactically and therapeutically 

5. activity against microorganisms varies

6. drug resistance may transfer to other bacteria

describe- activity is not directed at human metabolic process - target is the microorganism

• it has selective toxicity

• attacks the mircroorganism

• some targets are unique to the microorganism e.g. peptidoglycan synthesis

• other targets perform similar functions as those in eukaryotes but may have: significant structural differences leading to different active site conformations, different composition e.g. phospholipid, the cell may handle the drug differently, it may have a non-essential role in eukaryotic cells

• this does not mean that there will not be side effects to the patient

describe- activity is needed against several body sites

• one microorganism may infect a range of different body sites - why? travel in blood

• this is known as a focal infection- enters one part, then spreads by bloodstream

describe - used in large numbers of patients for a short period

• e.g. 3-5-7 days only

• for endocarditis, its used for a long period to ensure the infection has cleared

describe - used prophylactically and therapeutically 

• used to prevent infection

• used in infection

describe - activity against microorganisms varies

• one antibiotic can have multiple targets

• to do this, the drug target must be known

• when you don’t know what infection somebody has, you use an antibiotic with broad targeting e.g. tetracycline

Describe - drug resistance may transfer to other bacteria

• drug resistance may affect the recipient and may also disseminate clonally or by gene transfer to other bacteria 

• plasmids are extracellular loops of DNA

• some plasmids (conjugative plasmids) are capable of being passed between different bacterial species and even genera

• so plasmids can spread antibiotic resistance e.g. mating bridge

• bacteria can be spread by individuals (next card) & movement of people/travelling

how can disease be spread between individuals?

• communicable disease

• movement of people, travelling leading to resistant organisms

• for example, you carry your microflora with you

what are parts that make up spreading disease? like a cycle